Modulatory Roles of Human Adenovirus Type 3 in Extracellular Vesicle Development, Trafficking, and Inhibition

Date of Award

Spring 2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Qiana L. Matthews

Second Advisor

Brian Sims

Third Advisor

Ting Li

Abstract

Respiratory human adenovirus (HAdV) has been closely linked with acute respiratory infections such as pharyngitis and coryza (common cold). While some HAdV serotypes antagonize the immune system leading to meningitis, gastroenteritis, and acute hemorrhagic cystitis among other conditions, HAdV type 3 (HAdV3) predominantly infects the upper respiratory tract. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs), termed exosomes, may offer a mechanism by which certain viruses, enter cells via receptor-independent entry. EVs play a major role in both cellular homeostasis and intercellular communication under both healthy and pathological conditions. Firstly, in this study, we demonstrated that EVs isolated from HAdV3-infected A549 cells showed a significant increase in particle mean sizes, concentrations, and total EV content relative to EVs derived from uninfected cells. Infected EV cargo protein detection also upregulated levels of EV-associated classical, stress, and apoptotic response markers (CD63, TSG101, Caspase1, and IL-1β) levels mostly at the highest multiplicities of infection. EVs also expressed elevated levels of membrane trafficking protein Rab5, 7, and 35 at higher MOIs relative to uninfected. Secondly, we examined how inhibition of extracellular vesicle release and/or uptake would impact the exosome formation pathway. Using a constellation of improved EV experimental approaches, we evaluated the concentration-based cytotoxicity effects of pharmacological agents (ketoconazole, climbazole, and heparin) on human lung carcinoma (A549) cell viability. We investigated the effect of inhibitor dosages on exosome production and release. Analysis of exosome inhibition includes quantitative analysis and total protein expression of exosome release after pharmacological inhibition; we examined exosome protein level after inhibition. Selective inhibition of exosomes altered particle sizes, and heparin significantly reduced the total exosomes released. Climbazole and heparin undermined membrane-bound tetraspanin CD63 expression and significantly disrupted ALIX protein ( p ≤ 0.0001) and TSG101 ( p ≤ 0.001). Azoles and heparin also disrupt transmembrane trafficking by modulating Ras binding protein ( p ≤ 0.001). Lastly, we developed a cell-based assay to determine the effect of HAdV3 on the exosome inhibition process by azole and heparin derivatives. HAdV3-infected cells were treated with two concentrations of each inhibitor at different time points. HAdV3 activities led to increased total small RNA (sRNA), DNA, and exosome particle concentrations via particle tracking in the presence of climbazole and heparin relative to uninfected exosomes. In addition, there was an increased expression of classical markers such as ALIX, and tetraspanin (CD63), ( p <0.05) and upregulated transcription factor IRF8 in the presence of HAdV3 after 24 h of treatment. Whereas higher concentrations of climbazole and heparin sodium salt were found to inhibit total exosome protein ( p <0.001) and exo-RNA ( p <0.01) content even in the presence of HAdV3 relative to infected exosomes only. Activities of HAdV3 in the presence of selected inhibitors resulted in the positive regulation of exosome-related DNA damage/repair signaling proteins. Our findings suggested HAdV3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression, it further revealed that pharmacological inhibition of exosomes regulates the endocytic pathway and expression of the endosomal sorting complex required for transport mediators, suggesting climbazole and heparin as effective inhibitors of exosome synthesis and that HAdV3 may bolster inhibited exosome content and release while modulating certain activities of the mediators of the endosomal pathway.

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